Biomedical Engineering Reference
In-Depth Information
be introduced. This allows very i ne-tuned studies of, for example, ligand-receptor interactions and
protein function in general and has been described as “protein medicinal chemistry.”
A number of technologies have been developed to achieve this objective and it is now possible to
generate proteins containing, in principle, any functionality. In the following sections, we will focus
on two general methods that allow this: (1) unnatural mutagenesis, which allows the site-specii c
incorporation of unnatural amino acids into protein and (2) ligation-based strategies, which allows
semisynthesis of proteins and thereby the incorporation of a wide range of unnatural functionalities
into proteins.
4.3.2 U
NNATURAL
M
UTAGENESIS
In 1989, a biosynthetic
in vitro
method that allowed the site-specii c incorporation of unnatural
amino acids into proteins was introduced based on earlier work on nonsense suppression. The term
“nonsense suppression” refers to the use of stop (nonsense) codons and suppressor transfer RNA
(tRNA), which recognize stop codons. The method is based on the fact that only one of three stop
codons in the genetic code is necessary for the termination of protein synthesis and the two unused
stop codons can then be exploited for the introduction of unnatural amino acids.
The primary challenge in this technology is the generation of the modii ed suppressor tRNA with
the unnatural amino acid (Figure 4.8). Once generated, the aa-tRNA is recognized by the mRNA
carrying the specii c stop codon, whereby the unnatural amino acid is incorporated into the protein
at the specii c position. Based on this principle, two slightly different methodologies have been
developed for the site-specii c incorporation of unnatural amino acids: one method applies tRNAs
that are chemically aminoacylated with the unnatural amino acid of interest, and the aa-tRNA
is subsequently applied in an expression system to generate the protein of interest (Figure 4.8).
The other method employs the development of pairs of orthogonal tRNA and aminoacyl-tRNA
synthetases (aaRS), where the latter is developed so that it selectively recognizes aminoacylate an
unnatural amino acid.
In the chemical aminoacylation of tRNA, a dinucleotide is prepared by chemical synthesis and
subsequently aminoacylated with the unnatural amino acid of interest. The aa-tRNA is obtained
by the ligation of a truncated tRNA where a dinucleotide at the 3
-terminus is missing with the
prepared aminoacylated dinucleotide (Figure 4.8). If an
in vitro
expression system is used, the aa-
tRNA is simply added to the media, and when whole cell expression systems are used, the aa-tRNA
is injected into the cell. A particular attractive expression system for this methodology is
Xenopus
oocytes, which is generally used for electrophysiological studies of ion channels, receptors, and
transporters. The oocyte is coinjected with two RNA species: the modii ed mRNA encoding for the
target protein and the aa-tRNA chemically acylated with an unnatural amino acid. This coinjection
results in synthesis and surface expression of the target protein containing the unnatural amino acid
(Figure 4.8).
The methodology has been used to incorporate a large number of structurally diverse unnatural
amino acids, representing a large variety of functionalities, into proteins. In most cases the unnatu-
ral amino acids have been a-amino acids but also non-a-amino acids and most notably a-hydroxy
acids have been incorporated with the latter introducing an amide-to-ester mutation in the protein
backbone (Figure 4.9). These studies have shown that translation factors and the ribosome are com-
patible with many types of unnatural amino acids.
In studies of ligand-gated ion channels, such as nicotinic acetylcholine (nACh), g-aminobutyric
acid (GABA), and serotonin (5-HT
3
) receptors (see Chapters 12 and 14), the technology has proven
particularly valuable. These studies were pioneered by Dougherty and Lester, who have explored the
molecular details of the cation-p interaction between the quaternary ammonium group of acetylcholine
and aromatic residues in the nACh receptor; this was achieved by the site-specii c incorporation of
l uoro-substituted tyrosine and tryptophan residues, where the l uoro substituent gradually decreases
the ability of the aromatic moiety to interact in cation-p interactions. These studies have provided
unique details of acetylcholine interaction with subtypes of nACh receptors at the molecular level.
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